脾DNA的提取

1.Add 750μl Reagent B (50mM Tris [pH8],10mM EDTA,100mM NaCl,1% [w/v] SDS)and 50μl 100mg/ml proteinase K to each spleen.

2.Incubate overnight at 56ºC.

3.Gently shake to dissociate tissue.

4.transfer 200μl to eppendorf tube.

5.Add 50μl 5M sodium perchlorate.

6.Incubate 25 mins 65ºC.

7.Add 200μl phenol/chloroform.

8.Mix by inversion for about 2 mins.

9.Spin 13000rpm for 2 mins.

10.Transfer aqueous phase (top layer)to new eppendorf.

14 Add 500μl ice cold 100% ethanol.

15.Spool DNA into 500μl TE 10:0.l (if no visible DNA see below).

16 Allow to resuspend at 4ºC overnight.

If no visible DNA:

1.Spin 15 mins 13000rpm at ºC.

2.Remove liquid.

3.Add 500μl 70% ethanol.

4.Spin 5 mins 13000rpm at 4ºC.

5.Remove liquid and vac dry.

6.Add 500μl TE 10:0.1.